Pediatric Surgery International

, Volume 35, Issue 12, pp 1353–1361 | Cite as

An intra-amniotic injection of mesenchymal stem cells promotes lung maturity in a rat congenital diaphragmatic hernia model

  • Shohei TakayamaEmail author
  • Kohei Sakai
  • Shigehisa Fumino
  • Taizo Furukawa
  • Tsunao Kishida
  • Osam Mazda
  • Tatsuro Tajiri
Original Article



We aimed to evaluate the effect of human mesenchymal stem cells (hMSCs) on congenital diaphragmatic hernia (CDH) by intra-amniotic injection in a rat CDH model.


Nitrofen (100 mg) was administered to pregnant rats at E9.5. hMSCs (1.0 × 106) or PBS was injected into each amniotic cavity at E18, and fetuses were harvested at E21. The fetal lungs were classified into normal, CDH, and CDH-hMSCs groups. To determine the lung maturity, we assessed the alveolar histological structure by H&E and Weigert staining and the alveolar arteries by Elastica Van Gieson (EVG) staining. TTF-1, a marker of type II alveolar epithelial cells, was also evaluated by immunohistochemical staining and real-time reverse transcription polymerase chain reaction.


The survival rate after intra-amniotic injection was 72.1%. The CDH-hMSCs group had significantly more alveoli and secondary septa than the CDH group (p < 0.05). The CDH-hMSCs group had larger air spaces and thinner alveolar walls than the CDH group (p < 0.05). The medial and adventitial thickness of the pulmonary artery in the CDH-hMSCs group were significantly better (p < 0.001), and there were significantly fewer TTF-1-positive cells than in the CDH group (p < 0.001).


These results suggest that intra-amniotic injection of hMSCs has therapeutic potential for CDH.


Congenital diaphragmatic hernia Mesenchymal stem cell Intra-amniotic injection Fetal therapy Lung hypoplasia Nitrofen 



This work was supported in part by Grant-in-Aid for Exploratory Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (MEXT KAKENHI grant number 15K10926 [TF]). The English used in this manuscript was reviewed by Brian Quinn (Editor-in-Chief, Japan Medical Communication).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflicts of interest.


  1. 1.
    Chandrasekharan PK, Rawat M, Madappa R, Rothstein DH, Lakshminrusimha S (2017) Congenital diaphragmatic hernia—a review. Matern Health Neonatol Perinatol 3:6. CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Kimura O, Furukawa T, Higuchi K, Takeuchi Y, Fumino S, Aoi S et al (2013) Impact of our new protocol on the outcome of the neonates with congenital diaphragmatic hernia. Pediatr Surg Int 29(4):335–339. CrossRefPubMedGoogle Scholar
  3. 3.
    Ruano R, Yoshisaki CT, da Silva MM, Ceccon ME, Grasi MS, Tannuri U et al (2012) A randomized controlled trial of fetal endoscopic tracheal occlusion versus postnatal management of severe isolated congenital diaphragmatic hernia. Ultrasound Obstet Gynecol 39(1):20–27. CrossRefPubMedGoogle Scholar
  4. 4.
    Delens L, Jouret F, Detry O, Beguin Y, Krzesinski JM (2014) The role of mesenchymal stromal cells in solid organ transplantation. Rev Med Suisse 10(439):1540–1543 (1538) Google Scholar
  5. 5.
    Vanover M, Wang A, Farmer D (2017) Potential clinical applications of placental stem cells for use in fetal therapy of birth defects. Placenta 59:107–112. CrossRefPubMedGoogle Scholar
  6. 6.
    Moroncini G, Paolini C, Orlando F, Capelli C, Grieco A, Tonnini C et al (2018) Mesenchymal stromal cells from human umbilical cord prevent the development of lung fibrosis in immunocompetent mice. PLoS One 13(6):e0196048. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Li Y, Gu C, Xu W, Yan J, Xia Y, Ma Y et al (2014) Therapeutic effects of amniotic fluid-derived mesenchymal stromal cells on lung injury in rats with emphysema. Respir Res 15:120. CrossRefPubMedPubMedCentralGoogle Scholar
  8. 8.
    Ahn SY, Park WS, Kim YE, Sung DK, Sung SI, Ahn JY et al (2018) Vascular endothelial growth factor mediates the therapeutic efficacy of mesenchymal stem cell-derived extracellular vesicles against neonatal hyperoxic lung injury. Exp Mol Med 50(4):26. CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Furlani D, Ugurlucan M, Ong L, Bieback K, Pittermann E, Westien I et al (2009) Is the intravascular administration of mesenchymal stem cells safe? Mesenchymal stem cells and intravital microscopy. Microvasc Res 77(3):370–376. CrossRefPubMedGoogle Scholar
  10. 10.
    Kobayashi K, Lemke RP, Greer JJ (2001) Ultrasound measurements of fetal breathing movements in the rat. J Appl Physiol 91(1):316–320. CrossRefPubMedGoogle Scholar
  11. 11.
    Cooney TP, Thurlbeck WM (1982) The radial alveolar count method of Emery and Mithal a reappraisal 2–intrauterine and early postnatal lung growth. Thorax 37(8):580–583. CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Pua ZJ, Stonestreet BS, Cullen A, Shahsafaei A, Sadowska GB, Sunday ME (2005) Histochemical analyses of altered fetal lung development following single vs. multiple courses of antenatal steroids. J Histochem Cytochem 53(12):1469–1479. CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Roubliova XI, Deprest JA, Biard JM, Ophalvens L, Gallot D, Jani JC et al (2010) Morphologic changes and methodological issues in the rabbit experimental model for diaphragmatic hernia. Histol Histopathol 25(9):1105–1116. CrossRefPubMedGoogle Scholar
  14. 14.
    Yuniartha R, Alatas FS, Nagata K, Kuda M, Yanagi Y, Esumi G et al (2014) Therapeutic potential of mesenchymal stem cell transplantation in a nitrofen-induced congenital diaphragmatic hernia rat model. Pediatr Surg Int 30(9):907–914. CrossRefPubMedGoogle Scholar
  15. 15.
    Pederiva F, Ghionzoli M, Pierro A, De Coppi P, Tovar JA (2013) Amniotic fluid stem cells rescue both in vitro and in vivo growth, innervation, and motility in nitrofen-exposed hypoplastic rat lungs through paracrine effects. Cell Transplant 22(9):1683–1694. CrossRefPubMedGoogle Scholar
  16. 16.
    Di Bernardo J, Maiden MM, Hershenson MB, Kunisaki SM (2014) Amniotic fluid derived mesenchymal stromal cells augment fetal lung growth in a nitrofen explant model. J Pediatr Surg 49(6):859–865. CrossRefPubMedGoogle Scholar
  17. 17.
    Sakai K, Kimura O, Furukawa T, Fumino S, Higuchi K, Wakao J et al (2014) Prenatal administration of neuropeptide bombesin promotes lung development in a rat model of nitrofen-induced congenital diaphragmatic hernia. J Pediatr Surg 49(12):1749–1752. CrossRefPubMedGoogle Scholar
  18. 18.
    Montalva L, Zani A (2019) Assessment of the nitrofen model of congenital diaphragmatic hernia and of the dysregulated factors involved in pulmonary hypoplasia. Pediatr Surg Int 35(1):41–61. CrossRefPubMedGoogle Scholar
  19. 19.
    Kugler MC, Joyner AL, Loomis CA, Munger JS (2015) Sonic hedgehog signaling in the lung. From development to disease. Am J Respir Cell Mol Biol 52(1):1–13. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Umeda S, Miyagawa S, Fukushima S, Oda N, Saito A, Sakai Y et al (2016) Enhanced pulmonary vascular and alveolar development via prenatal administration of a slow-release synthetic prostacyclin agonist in rat fetal lung hypoplasia. PLoS One 11(8):e0161334. CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Boucherat O, Benachi A, Barlier-Mur AM, Franco-Montoya ML, Martinovic J, Thebaud B et al (2007) Decreased lung fibroblast growth factor 18 and elastin in human congenital diaphragmatic hernia and animal models. Am J Respir Crit Care Med 175(10):1066–1077. CrossRefPubMedGoogle Scholar
  22. 22.
    Shehata SMK, Sharma HS, van der Staak FH, van de Kaa-Hulsbergen C, Mooi WJ, Tibboel D (2000) Remodeling of pulmonary arteries in human congenital diaphragmatic hernia with or without extracorporeal membrane oxygenation. J Pediatr Surg 35(2):208–215. CrossRefPubMedGoogle Scholar
  23. 23.
    Herriges M, Morrisey EE (2014) Lung development: orchestrating the generation and regeneration of a complex organ. Development 141(3):502–513. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
    Takayasu H, Nakazawa N, Montedonico S, Sugimoto K, Sato H, Puri P (2007) Impaired alveolar epithelial cell differentiation in the hypoplastic lung in nitrofen-induced congenital diaphragmatic hernia. Pediatr Surg Int 23(5):405–410. CrossRefPubMedGoogle Scholar
  25. 25.
    Di Bernardo J, Maiden MM, Jiang G, Hershenson MB, Kunisaki SM (2014) Paracrine regulation of fetal lung morphogenesis using human placenta-derived mesenchymal stromal cells. J Surg Res 190(1):255–263. CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Shohei Takayama
    • 1
    • 2
    Email author
  • Kohei Sakai
    • 1
  • Shigehisa Fumino
    • 1
  • Taizo Furukawa
    • 1
  • Tsunao Kishida
    • 2
  • Osam Mazda
    • 2
  • Tatsuro Tajiri
    • 1
  1. 1.Department of Pediatric SurgeryKyoto Prefectural University of MedicineKyotoJapan
  2. 2.Department of ImmunologyKyoto Prefectural University of MedicineKyotoJapan

Personalised recommendations